Our in vivo studies in mice indicate that histone deacetylase inhibitors (HDACi) enhance the numbers and/or suppressive function of regulatory T cells (Tregs), through effects on Foxp3+ cell production, and histone and FoxpS acetylation. We propose to explore aspects of HDACi use with relevance to experimental models of transplant rejection and autoimmunity. Aim #1: Does the balance between acetylation and deacetylation regulate Treg-dependent functions? Our preliminary studies indicate that Foxp3+ CD4+CD25+ Tregs have considerable HDAC functional activity, and that HDACi therapy can enhance Treg functions in vitro and in vivo. Our proposed studies will determine if HDACi therapy: 1.1) increases Treg numbers by affecting thymic production, peripheral conversion or increased lifespan; 1.2) increases Treg suppressive functions; 1.3) has effects on non-Tregs that may explain the therapeutic effects observed in vivo; 1.4) depends on the use of a particular agent or is a general action of HDACi on Tregs; 1.5) is enhanced by combination with immunosuppression; and 1.6) is enhanced by combination with DNA methyltransferase inhibitors? Aim #2: Which HDACs are involved and what are they affecting in Tregs? Based on our initial data of HDACi affecting the size and the function of the Foxp3+ Treg pool, and data from our analysis of HDAC9- deficient mice showing increased numbers and suppressive function of Foxp3+ Tregs, we will investigate the following: 2.1) does the prototypic class II HDAC, HDAC9, control Treg numbers, gene expression and function; 2.2) how is HDAC9 regulated, and is the catalytic activity of HDAC9 required for its effects in Tregs; 2.3) do class I HDACs also contribute to the regulation of Treg function; and 2.4) is acetylation of Foxp3 itself a key regulator of Treg functions? This project will complement projects #1 and #2 by testing in vivo key concepts and insights developed by the detailed biochemical studies of the FoxpS complex in project #1, and the various structure/function relationships for FoxpS arising from project #2. Project 3 will also rely on access to appropriate transgenic and mutant mice developed by the Transgenic Core. We anticipate that our studies will have two main benefits for the broad scientific community and ultimately for clinical care. First, they will likely provide the basic insights into how immune monitoring can move beyond the current state of was FoxpS detected or not? i.e. important new criteria for evaluation of the level of activation and functional competency of Foxp3+ Tregs should be generated by our work. Second, they are likely to provide the basis for the rational use of HDACi for pharmacologic enhancement of Treg functions in vivo, and will likely also suggest important new targets for future therapeutic targeting (e.g. HDAC9-selective blockade).